Much attention in the proteomics and related research fields is devoted to the study of phosphorylation and other forms of post-translational modification of proteins. In such studies, mass spectrometers are commonly utilized to identify and sequence modified peptides. Data-dependent neutral loss analysis is a particularly useful technique for this purpose. In data-dependent neutral loss analysis, a survey (full MS) scan is initially performed to automatically and rapidly select one or more precursor ions of interest, for example, to select those ion species corresponding to the N most intense peaks in the MS spectrum. MS/MS analysis is then performed on the identified precursor ion(s), and the resultant spectra are analyzed to determine if any of the product ion species have masses that differ from the corresponding precursor ion mass by a specified amount (the neutral loss) which corresponds to the loss of a phosphate group or other modification. If it is determined that the mass of one or more product ion species differs from its precursor by the specified neutral loss amount, then a subsequent stage of fragmentation and analysis (MS3) is performed on the product ion in order to generate additional mass spectral information that can be used to identify the site of modification.
Because data-dependent neutral loss analysis necessarily involves a relatively large number of mass analysis scans (e.g., one mass analysis cycle may involve a full MS scan and several MS/MS and MS3 scans), the technique's implementation has been largely limited to ion trap mass spectrometers, which have the ability to execute the scans in rapid fashion. Typically, the ion trap mass spectrometer is configured to trigger the MS3 scan on a product ion having a “nominal” neutral loss value rounded to the nearest integer value (e.g., 98 for phosphate). This approach suffers from low selectivity, however, because many molecular moieties can produce neutral losses having the same nominal neutral loss value. In this manner, many MS3 scans will be performed unnecessarily, which may overburden the data system and reduce sample throughput. While it is desirable to use a more precise neutral loss value to trigger MS3 to improve selectivity, such precision is generally beyond the capability of commercially-available ion traps when operated at normal scan rates.
Various references (see, e.g., Bogdanov et al., “Proteomics by FTICR Mass Spectrometry: Top Down and Bottom Up”, Mass Spectrometry Reviews, Vol. 25, pp. 168-200 (2005)) discuss the use of mass analyzers capable of generating high-resolution, high mass accuracy mass spectral data, such as the Fourier Transform/Ion Cyclotron Resonance (FTICR) and Orbitrap analyzers, for identification and characterization of peptides and proteins. The accurate mass information produced by mass analyzers of this type facilitates the identification of known compounds and determination of elemental composition for unknowns. High resolution analysis allows isobaric compounds to be separated from one another or from chemical noise. However, the relatively slow mass spectra acquisition rates of high-resolution, high mass accuracy mass analyzers limit sample throughput and may be insufficient for execution of multiple MSn cycles on a chromatographic timescale.